Ontogenic Expression Profiles of Gonadotropins (fshb and lhb) and Growth Hormone (gh) During Sexual Differentiation and Puberty Onset in Female Zebrafish

In the zebrafish model, the ontogenic expression profiles of all pituitary hormones have been reported except gonadotropins, partly because they are not supposed to be expressed in the embryonic stage. The spatiotemporal expression patterns of gonadotropins, namely follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh), in this species therefore remain largely unknown. As the master hormones controlling reproduction, the information on this issue would be valuable for understanding the roles of gonadotropins in early sexual development. Using double-colored in situ hybridization and real-time qPCR, this study was undertaken to analyze the ontogenic expression patterns of FSHbeta (fshb) and LHbeta (lhb) subunits in the zebrafish pituitary with particular emphasis on the stage of sexual differentiation (~25-30 dpf) and puberty onset (~45 dpf). As a control, growth hormone (gh) was also examined throughout the study. The zebrafish were collected at different time points of early development including 4, 5, 6, 8, 10, 13, 16, 19, 22, 25, 28, 38, 48 and 53 days postfertilization (dpf). The head of each fish including the brain and pituitary was sampled for double-colored fluorescent in situ hybridization (FISH) analysis, whereas the body was fixed for histological examination of sex and gonadal developmental stage. Our results showed that the expression of fshb started much earlier than that of lhb with its mRNA signal detectable (~2-3 cells/pituitary) shortly after hatching (4 dpf). In contrast, lhb expression became detectable much later at the time of sex differentiation (~25 dpf). In female zebrafish, the first morphological sign for puberty is the first wave of follicle transition from the primary growth (PG) to previtellogenic stage (PV), which occurs around 45 dpf and is marked by the appearance of cortical alveoli in the oocytes. Interestingly, the number of lhb-expressing cells was very low (~5-6 cells/pituitary) before this transition but increased dramatically during and after the transition. In contrast, the expression of fshb was abundant before puberty with only a slight increase in cell number during puberty onset. The increased expression of fshb and lhb at puberty was also supported by real-time qPCR analysis at single pituitary level. Interestingly, the fshb-expressing cells changed their spatial distribution significantly during puberty from predominantly peripheral to central location. < truncated - database field length exceeded>